This invention is directed to the use of a single, substantially pure, polymethine dye for the cytological preparation of a fixed biopsy specimen of hematopoietic origin. More specifically, this invention relates to the use of a single polymethine dye for differentiating, identifying and enumerating cells among a plurality of cells of hematopoietic origins selected from the group consisting of blood cells, bone marrow cells and lymph node cells. The polymethine dyes of this invention can be used to stain a plurality of lymphocytes of hematopoietic origin by treating a fixed biopsy specimen with a solution of the polymethine dye and subsequently identifying and enumerating the stained cells under dark field illumination. The polymethine dyes particularly useful for purposes of this invention are available as Astrazon Violet F.sub.3 RL (BV 21) and Astrazon Red Violet FRR. The stained lymphocyte subpopulations prepared in accordance with this invention have excellent color stability, are remarkably clear with respect to cellular detail and have brilliant cell structure.
In general, there are various methods for counting and classifying leukocytes. For example, the cytotechnician microscopically views a biopsy specimen prepared on an ordinary microscope slide that has been stained with one of the Romanowsky stains. The cytotechnician examines the stained leukocytes and classifies them according to cell type. This method, however, is time consuming and has the disadvantage of not being reliable with respect to counting and classifying the less abundant cells such as the monocytes, eosinophils, basophils, etc. In a typical automated system, a biopsy specimen is stained in the usual manner with a Romanowsky stain and then scanned mechanically under a microscope provided with an electronic image tube. When the leukocyte comes in view of the image tube, the slide is stopped. An image analyzing computer connected to the image tube classifies the leukocyte according to its cell profile and cytoplasm color. Here also, the system suffers from the disadvantage of being too time consuming and costly.
In comparison, the use of the polymethine dyes of this invention is an improvement over the Romanowsky stains even though mixtures of dyes are still the basis for morphological hematology and are being used for the identification of cells. The Romanowsky-type stains are commonly known to contain Methylene Blue and eosin. The mixture of dyes produces the Romanowsky effect defined as a condition wherein the coloration of the cell components results from the combined action of the mixture of dyes, and cannot be produced by either dye acting alone. The state of the art as presently developed, fails to teach the use of a single, substantially pure, commercially available dye capable of panoptically and metachromatically staining a fixed biopsy specimen. The polymethine dyes of this invention, however, are capable of staining cells of hematopoietic origin comprising blood cells, bone marrow cells or lymph node cells which permits the differentiation, enumeration and identification of the individual cells, i.e. the subpopulations of lymphocytes including the abnormal or malignant cells.
In recent years, the identification of lymphocytes and their various subpopulations has become more important and more interesting to biomedical researchers especially for diagnostic purposes. More particularly, the various subpopulations of lymphocytes have been better defined and described and their functions in the immune system have become more clearly delineated. More specifically, the important subpopulations include the B-cells, T-helper cells, T-suppressor cells, and Natural Killer (NK) cells. The B-cells are believed to be bursa derived and are involved in the synthesis of circulating immunoglobulins. According to recent evidence, the T-helper and T-suppressor cells are involved in the modulation of the immune response and in the regulation of erythropoiesis. More important, the Natural Killer cells represent the body's first line of defense against malignancy, since these cells are directly cytotoxic to any foreign cells, and do not require the mediation of complement to effect their lysis.
Within the past several years, however, the function and complexity of the T-helper and T-suppressor cell populations have received an increasing amount of attention. Currently, it is believed that the HTLV-I virus is the specific etiologic agent for adult T-cell leukemia. This virus specifically attacks the T-helper cell by entering the genetic material and "immortalizes" the cell, transforming it into a large leukemic lymphoblast with striking convolutions and indentations of the nucleus and containing a large nucleolus. Presumably, the HTLV-I virus particles replicate within the T-helper cell and are shed into the circulation system to infect other T-helper cells.
In contrast to the "immortalization" of the T-helper cell in the HTLV-I virus infestation, the HTLV-III virus infects the T-helper cells specifically and in so doing destroys the cells. It is important to note that the HTLV-III virus is believed to be the specific causative agent of the acquired immune deficiency syndrome (AIDS). In this disorder (AIDS), it was found that the number of T-helper cells is greatly diminished because of the lethal effects of the virus, leading to a reversal of the ratio between the T-helper cell and the T-suppressor cell. Morevoer, information about the antibodies to the HTLV-III virus, and the ratio of the T-helper to the T-suppressor cells is a valuable diagnostic test for determining the presence of HTLV III infection, and also for confirming the diagnosis of AIDS.
Further, the identification and enumeration of the various lymphocyte subpopulations continues to be increasingly important in other disorders of presumed immune origin, such as collagen diseases and the allergic disorders as well as in the investigation of malignancies and the role of the immune system in the defense against malignancies. Studies have also demonstrated that there are abnormalities in the T-cell subset populations in patients with malignancies treated with chemotherapy. Whether or not disturbances in these immune mechanisms and in the mechanisms of immune surveilance contribute to the development of malignancies and to the ultimate development of acute leukemia in patients treated with chemotherapy is a provocative and as of now an unanswered question.
The prior art teaches that the identification of the various lymphocyte subpopulations can be achieved, for the most part, with the use of rosette formations using either untreated sheep red blood cells for T-cells and treated sheep red blooded cells for B-cells. Another more common method is the use of monoclonal antibodies direct against specific surface antigens on the various lymphocytes. Although these antibodies are expensive to use and require a tedious methodology, the monoclonal antibodies are highly specific. However, because of their complexity, particularly with respect to sample preparation, the monoclonal antibodies have not been in widespread use in the smaller hospitals, laboratories, physicians offices, clinics and the like.
Initially, Kass taught that the identification of T-cells and B-cells and in several cases other lymphocyte subpopulations could be accomplished by using a single organic dyestuff; see U.S. Pat. Nos. issued to Dr. L. Kass Nos. 4,400,370 and 4,500, 509 and U.S. patent application Ser. Nos. 129,680 filed Mar. 12, 1980; 604,741 filed Apr. 27, 1984; 604,869 filed Apr. 27, 1984; and 765,348 filed Aug. 13, 1985. The Kass patents disclose that Basic Orange 21 is a supravital stain and therefore must be used on living blood cells in a liquid suspension viewed in the presence of absorbance or fluorescent light. The Basic Blue 41 dye, however, must be used on a fixed biopsy specimen. Here the lymphocyte subpopulations were identified on the basis of the difference in the intensity of staining and the caliber of the nuclear chromatin strands in the nucleus, as well as the presence of metachromatically staining granules in the cytoplasm. By using the Basic Blue 41 stain, the lymphocyte subpopulations could be identified under absorbance light, since the cells do not fluoresce nor do they demonstrate differential coloration when viewed under dark field illumination. However, by using still another stain, i. e. Basic Blue 141, the B-cell and T-cell subpopulations displayed differential staining in a fixed biopsy specimen but the cells did not fluoresce, nor did they exhibit any differential coloration when viewed under a dark field illumination.
Accordingly, it is an object of this invention to provide a single, substantially pure, polymethine dye for staining fixed cells of hematopoietic origin for the identification and enumeration of lymphocyte subpopulations.
It is another object of this invention to provide a single polymethine dye capable of staining lymphocyte subpopulations of hematopoietic origin which can be viewed under dark field illumination for purposes of identification and enumeration.
It is another object of this invention to provide novel stained cells of hematopoietic origin, and more particularly stained lymphocyte subpopulations of hematopoietic origin having individual color characteristics which permit the differentiation, identification and enumeration of said cells.
It is a further object of this invention to provide a method of staining a plurality of cells of hematopoietic origin by reacting a single polymethine dye with a fixed biopsy specimen to obtain a plurality of stained cells having individual color characteristics which permit the differentiation, identification and enumeration of said stained cells under dark field illumination. These and other objects of the invention will become apparent from a further more detailed description of the invention as follows.